1. Field of the Invention
This invention pertains to a method of separating free from bound fractions in an immunoassay procedure and to a novel immunochemical composite for use therein.
2. Description of the Prior Art
Solid phase radioimmunoassay (RIA) has become popular because the system in which both the antigen-antibody reaction and the separation of free and bound antigen can be achieved in a single step results not only in a simple and rapid RIA, but also eliminates a number of handling and other errors which are inherent in other separation techniques. K. J. Catt, H. D. Niall, and G. W. Tregear, Biochem. J., 100: 31c (1966), originally used as solid phase materials powdered polymers bearing reactive thiocyanate groups (--N.dbd.C.dbd.S) capable of forming covalent linkages with antibodies. Antibodies coupled to cyanogen-bromide-activated dextran and cellulose particles came into vogue as a result of the work of Wide, Porath, and Axen [L. Wide and J. Porath, Biochem. Biophys. Acta, 130: 257 (1966), R. Axen, J. Porath and S. Ernback, Nature (Lond.), 214: 1302 (1967), and L. Wide, Acta Endocrinol. (Copenhagen) Suppl. No. 142: 207 (1969)]. Alternatively, C. Ternynck and S. Azrameas, F. E. B. S. Letts., 23: 24 (1972), used gluaraldehyde as a two-step bifunctional reactant to couple antibodies to the amide groups of polyacrylamide.
It has been well established that the efficiency of affinity adsorbents increase considerably when hydrocarbon spacers are introduced to separate the ligand from the solid matrix [P. Cuatrecasas, M. Wilcheck, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S., 61: 636 (1968)]. The spacer is thought to increase the flexibility and mobility of the ligand allowing unhindered access of the protein to the ligand. Armed with this knowledge, C. L. Cambiaso, A. Gossinet, J. P. Vaerman, and J. F. Heremans, Immunochem., 12: 273 (1975), coupled gamma globulin fractions to glutaraldehyde-activated amino-hexyl derivatives of sepharose-4.beta., as shown in FIG. 1, and showed this procedure to produce useful immunosorbents. ##STR2##